Carotenoids are a large class of yellow, orange and red lipophilic structures synthesized by all photosynthetic organisms. In plants, carotenoids play multiple roles such as photosynthetic light harvesting, protection against light and heat stress, and as precursors to hormones that mediate stress and developmental signalling. Carotenoid antioxidants increase heat and light stress tolerance by protecting membranes from reactive oxygen species (ROS) and lipid peroxidation. Concentrated in fibrillar plastoglobuli of fruit chromoplasts, carotenoids are attractants to animals that serve as plant seed distributors. Certain carotenoids in the endosperm tissue provide nutritional value and have been targets for improvement, especially in cereal crops of the grass family.
The biosynthetic pathway of carotenoids occurs in plastids where the lipophilic carotenoids accumulate in envelope/thylakoid membranes and plastoglobuli. Carotenoids are synthesized in both light and dark-grown tissues, such as leaves, endosperm, and roots. In the dark, leaf tissues develop etioplasts with rudimentary prolamellar bodies, which are the precursors for thylakoids and support low levels of carotenoid biosynthesis. In the light, leaves turn green due to development of highly specialized chloroplasts filled with complex photosynthetic systems. Carotenoids are distributed differently in etioplasts and chloroplasts that might require differential localization of their biosynthetic enzymes as well.
Phytoene synthase (PSY) catalyzes the committed step to carotenoid biosynthesis and is a key target for pathway engineering. There are up to three PSY isozymes in evolutionarily distant plants, including all the major food staples in the grasses and other crops of agronomic importance. Different PSY isozymes mediate carotenogenesis in particular tissues, in response to developmental and physiological signals. Allele-specific variation accounts for yellow endosperm maize and yellow rooted cassava.
The core carotenoid biosynthetic pathway consists of about 10 enzymes. However, the location of the biosynthetic pathway as a complete entity for controlling the unique spatial distribution of carotenoids is unknown. Moreover, this pathway must respond to environmental and developmental signals to link photomorphogenesis, photoprotection, and stress responses with location-specific carotenoid synthesis and degradation. It has long been desired to understand the nature of this dynamic pathway landscape and how isozymes and allelic variants fit into the picture.
According to recent proteomic studies on Arabidopsis chloroplasts, many of the carotenoid biosynthetic pathway enzymes are exclusively localized to envelope membranes. Only a few carotenoid enzymes are found in thylakoids: xanthophyll cycle enzymes and phytoene desaturase (PDS). For example, in pepper fruit chromoplasts, most carotenoid enzymes are localized to plastoglobuli. In maize proteomic studies, the only carotenoid enzymes detected were PDS and □-carotene desaturase (ZDS) that were respectively found in membrane fractions of bundle sheath and mesophyll cells. Carotenoids are found in both cell types, yet other carotenoid biosynthetic enzymes were undetectable. Chloroplast suborganellar localization of the key pathway enzyme, PSY, has yet to be detected by proteomic analysis.
As a result of this invention, it has been discovered that PSY isozymes differ in chloroplast suborganellar localization and that overexpression of naturally occurring allelic variants produces striking differences in localization and profound effects on chloroplast architecture.